Observation apparatus and observation method

ABSTRACT

An observation apparatus in this embodiment includes a light source configured to irradiate an observation target with light, and a processing unit configured to generate an image based on Rayleigh scattered light derived from χ (3)  included in light obtained from the observation target.

The contents of the following Japanese patent applications areincorporated herein by reference:

NO. 2016-107936 filed in JP on May 30, 2016.

NO. PCT/JP2017/019977 filed in JP on May 29, 2017.

BACKGROUND 1. Technical Field

The present invention relates to an observation apparatus and anobservation method.

2. Related Art

In the prior art, in a pathological diagnosis through which apathologist makes a diagnosis of cancer or the like, an observationaldiagnosis is made using a bright field microscope after a tissue sectionis prepared and stained. The problem involved in such a technique isthat it takes much time to prepare a sample. Therefore, a tool isrequired for making a quick judgment, for example, whether the tissuehas turned to cancer without preparing a section in an observation of atissue cut from a patient or in an intraoperative tissue diagnosisduring open abdominal surgery, endoscopic surgery or the like. Toachieve the objective, it is required that the microscope not be oftransmission type but of reflection type, and an unstained imagingtechnique be used which has the sectioning capability capable ofobtaining a cross-sectional image of any position in the Z-direction ina plane perpendicular to the optical axis direction. For the unstainedimaging technique, the microscope utilizing the coherent interaction isknown (see Patent Document 1, for example). However, for the microscopeutilizing the coherent interaction, when it is of reflection type,sufficient signal intensity may not be obtained and a side wall of acell, for example, cannot be observed.

PRIOR ART DOCUMENT

[Patent Document] Patent Document 1: Japanese Patent ApplicationPublication No. 2005-62155

SUMMARY

According to the first aspect of the present invention, an observationapparatus includes a light source configured to irradiate an observationtarget with light, and a processing unit configured to generate an imagebased on Rayleigh scattered light derived from χ⁽³⁾ included in lightobtained from the observation target.

According to the second aspect of the present invention, an observationapparatus includes a light source configured to irradiate an observationtarget with light, and a detecting unit configured to at least partiallyblock light derived from χ⁽¹⁾ and detect Rayleigh scattered lightderived from χ⁽³⁾, where the Rayleigh scattered light derived from χ⁽³⁾and the light derived from χ⁽¹⁾ are included in light obtained from theobservation target.

According to the third aspect of the present invention, an observationmethod includes generating an image based on Rayleigh scattered lightderived from χ⁽³⁾ included in light obtained from an observation targetirradiated with light from a light source.

According to the fourth aspect of the present invention, an observationmethod includes at least partially blocking light derived from χ⁽¹⁾ anddetecting Rayleigh scattered light derived from χ⁽³⁾, where the Rayleighscattered light derived from χ⁽³⁾ and the light derived from χ⁽¹⁾ areincluded in light obtained from an observation target irradiated withlight from a light source.

According to the fifth aspect of the present invention, an observationapparatus includes a light source configured to irradiate an observationtarget with excitation light; at least one light splitting unit arrangedon an optical path of light obtained from the observation target andconfigured to split the light into beams of light; at least two mirrorscapable of adjusting an optical path length difference between the beamsof light from the light splitting unit; and a detector configured todetect Rayleigh scattered light derived from χ⁽³⁾ included in beams oflight reflected by the at least two mirrors, wherein the beams of lightfrom the light splitting unit are combined after reflected by the atleast two mirrors, and the detector detects the Rayleigh scattered lightderived from χ⁽³⁾ included in combined light.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an observation apparatus 100.

FIG. 2 is an illustrative view in which wavelength spectra are compared.

FIG. 3 is a diagram representing a process in which light having afrequency of ω² is emitted after light having a frequency of ω¹ enters.

FIG. 4 is a graph showing dependency of a theoretical signal intensityof light on an optical path difference length.

FIG. 5 is a graph showing dependency of an actually measured signalintensity of light on an optical path difference length.

FIG. 6 is a schematic view of an observation apparatus 200.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, (some) embodiment(s) of the present invention will bedescribed. The embodiment(s) do(es) not limit the invention according tothe claims, and all the combinations of the features described in theembodiment(s) are not necessarily essential to means provided by aspectsof the invention.

FIG. 1 is a schematic view of an observation apparatus 100. Theobservation apparatus 100 includes a confocal optical system 101, adetecting unit 102, and a controlling unit 104.

The confocal optical system 101 includes a laser light source 110,lenses 121, 123, 125, and 127, pinholes 131 and 133, a half-silveredmirror 140, a bandpass filter 145, a pair of X-Y scanning mirrors 150, ascanning driving unit 156, and an objective lens 160. The confocaloptical system 101 is an epi-illumination optical system, and theobjective lens 160 of the confocal optical system 101 is broughtadjacent to the observation target 10 when the observation target 10 isobserved.

The laser light source 110 emits narrowband CW laser light as excitationlight. A light source which is commonly used for a Raman microscope isused as the laser light source 110, and the laser light preferably has ashort wavelength, for example approximately 488 nm in the visibleregion.

The lens 121, which is arranged on an optical path of the observationapparatus 100 at such a position as to receive the excitation lightemitted from the laser light source 110, converges the excitation lighton a circular aperture of the pinhole 131 arranged adjacent thereto. Thelens 123, which is arranged on the optical path of the observationapparatus 100 at a position adjacent to the pinhole 131, collimates theexcitation light from the laser light source 110 which has passedthrough the circular aperture of the pinhole 131.

The half-silvered mirror 140, which is arranged at a predetermined angleon the optical path of the observation apparatus 100, transmits at leasta part of the collimated excitation light from the lens 123. Thehalf-silvered mirror 140 also reflects at least a part of collimatedback-reflected light from the pair of X-Y scanning mirrors 150, changingthe optical path of the reflected part of collimated back-reflectedlight.

The pair of X-Y scanning mirrors 150, which is arranged on the opticalpath of the observation apparatus 100, reflects the collimatedexcitation light from the half-silvered mirror 140, changing the opticalpath of the collimated excitation light. The pair of X-Y scanningmirrors 150 also reflects back-reflected light from the observationtarget 10 which has passed through and has been collimated by theobjective lens 160, directing the back-reflected light in the reversedirection along the optical path of the excitation light from thehalf-silvered mirror 140. The pair of X-Y scanning mirrors 150 isresonance-type galvano mirrors, for example, and has X-Y scanningmirrors 151 and 153 which rock about the respective axes the directionsof which are different from each other. The scanning driving unit 156drives the pair of X-Y scanning mirrors 151 and 153 to change theoptical path of the light entering the pair of X-Y scanning mirrors 150two-dimensionally and in the direction crossing the optical axis.

The objective lens 160, which is arranged on the optical path of theobservation apparatus 100 at such a position as to receive thecollimated excitation light from the pair of X-Y scanning mirrors 150,converges the excitation light on a certain point on the observationtarget 10 arranged adjacent thereto. The objective lens 160 alsocollimates the back-reflected light from the observation target 10.

The bandpass filter 145, which is arranged on the optical path of theobservation apparatus 100, receives the back-reflected light from theobservation target 10 reflected by the half-silvered mirror 140 andtransmits light of a predetermined wavelength band and filters out lightof the other wavelength bands. To be specific, as described below indetail, the bandpass filter 145 receives the light from the observationtarget 10, and transmits light derived from χ⁽¹⁾, and transmits Rayleighscattered light and filters out light other than the Rayleigh scatteredlight, where the Rayleigh scattered light and the light other than theRayleigh scattered light are included in the light derived from χ⁽³⁾. Inthis way, Raman scattered light or intrinsic fluorescence, which isincluded in light from the observation target 10, is prevented fromentering the detecting unit 102.

The lens 125, which is arranged on the optical path of the observationapparatus 100, converges the light from the observation target 10 whichhas been transmitted through the bandpass filter 145 on a circularaperture on the pinhole 133 arranged adjacent thereto. The lens 127,which is arranged on the optical path of the observation apparatus 100at a position adjacent to the pinhole 133, collimates the light from theobservation target 10 which has passed through the circular aperture ofthe pinhole 133 and directs the collimated light to the inside of thedetecting unit 102 arranged adjacent thereto.

As described above, in the confocal optical system 101, the position ofthe circular aperture of the pinhole 131 and the certain point on theobservation target 10 that is a focus position of the objective lens 160are conjugate with each other. Similarly, the certain point on theobservation target 10 that is the focus position of the objective lens160 and the position of the circular aperture of the pinhole 133 areconjugate with each other.

The detecting unit 102 includes a blocking unit 103, a pinhole 135, adetector 190, and an output unit 191. The detecting unit 102 is arrangedadjacent to the confocal optical system 101 to receive the light fromthe observation target 10, which has passed through and has beencollimated by the lens 127 of the confocal optical system 101.

The blocking unit 103 has a configuration that is the same as that of aMichelson interferometer, for example, and has a beam splitter 170, apair of mirrors 180, and a vibration driving unit 186. The blocking unit103 at least partially blocks some of a plurality of light componentsincluded in the light from the observation target 10. Note that theconfiguration of the blocking unit 103 may be the same as theconfiguration of, for example, a Mach-Zehnder interferometer which ispartially different from the one shown in the figure, that is, may be aconfiguration which has two beam splitters 170.

The beam splitter 170, which is arranged on the optical path of theobservation apparatus 100, splits the collimated light from the confocaloptical system 101 into two beams of light with the same signalintensity. The beam splitter 170 also combines two collimated beams oflight reflected by the pair of mirrors 180. Note that the beam splitter170 is one example of a light splitting unit.

The pair of mirrors 180, which is arranged on the optical path of theobservation apparatus 100, are a pair of mirrors which can adjust anoptical path length difference between the two beams of light from thebeam splitter 170. The pair of mirrors 180 has a movable mirror 181 anda stationary mirror 183. The vibration driving unit 186 is for example,a piezo element that converts voltage applied on a piezoelectric bodyinto force, and changes the position of the movable mirror 181 along theoptical path. The vibration driving unit 186 also causes the movablemirror 181 to vibrate about a predetermined center along the opticalpath.

The pinhole 135, which is arranged on the optical path of theobservation apparatus 100, is located such that the circular aperture ofthe pinhole 135 partially stops the collimated light which has not beenblocked by the blocking unit 103. The detector 190, which is forexample, a photomultiplier and is arranged adjacent to the pinhole 135on the optical path of the observation apparatus 100, detects thecollimated light which has not been blocked by the blocking unit 103 andhas not been stopped by the circular aperture of the pinhole 135. Notethat the detector 190 is one example of a detector.

The output unit 191, which is for example, a processing apparatus havinga display and is electrically connected to the detector 190, displays anobservation image of the observation target 10 generated from the lightdetected by the detector 190 to an observer. Note that the output unit191 is one example of a processing unit.

As described above, the position of the circular aperture of the pinhole133 in the confocal optical system 101 and the position of the circularaperture of the pinhole 135 in the detecting unit 102 are conjugate witheach other. Therefore, in the entire observation apparatus 100, acertain point on the observation target 10 that is a focus position ofthe objective lens 160 and the position of the circular aperture of thepinhole 135 arranged in front of the detector 190 are conjugate witheach other. With the configuration, unnecessary scattered light fromsurroundings can be eliminated by a plurality of pinholes and only thelight from the focused position on the observation target 10 can bedetected at high contrast and high resolution.

The controlling unit 104, which is electrically connected to theconfocal optical system 101 and the detecting unit 102, controls drivingof the scanning driving unit 156 in the confocal optical system 101 anddriving of the vibration driving unit 186 in the detecting unit 102.

The entire configuration of the observation apparatus 100 is describedas above. Now the details of the light obtained from the observationtarget 10 are described using FIG. 2.

FIG. 2 is an illustrative view in which wavelength spectra are compared.(A) of FIG. 2 schematically shows a wavelength spectrum of theexcitation light emitted from the laser light source 110, (B) of FIG. 2schematically shows a wavelength spectrum of coherent light derived fromχ⁽¹⁾ included in the light obtained from the observation target 10irradiated with the excitation light, and (C) of FIG. 2 schematicallyshows a wavelength spectrum of incoherent light derived from χ⁽³⁾included in the light obtained from the observation target 10 irradiatedwith the excitation light, respectively.

The light obtained from the observation target 10 irradiated with theexcitation light includes light derived from χ⁽³⁾ and light derived fromχ⁽¹⁾. χ⁽¹⁾ means the linear susceptibility. The light derived from χ⁽¹⁾refers to the light originated in the term proportional to χ⁽¹⁾ when thepolarization induced by electric field of excitation light is expandedin a power series. On the other hand, χ⁽³⁾ means the third-ordernon-linear susceptibility. The light derived from χ⁽³⁾ refers to thelight originated in the term proportional to χ⁽³⁾ when the polarizationinduced by electric field of the excitation light is expanded in a powerseries.

For the light derived from χ⁽¹⁾, there is coherence among scatteredbeams of light, and all of a center wavelength, a phase, and a linewidth are the same as those of the excitation light. As understood fromthe comparison between (A) and (B) of FIG. 2, the spectral width of thelight derived from χ⁽¹⁾ is the same as the line width of the excitationlight from the laser light source 110. The light derived from χ⁽¹⁾ iscoherent light. The generated light derived from χ⁽¹⁾ has a clear phase.When beams of light each having a clear phase interfere mutually, theconstructive interference which means the waves add each other occursonly in certain directions and the destructive interference which meansthe waves cancel each other occurs in the other directions. For thisreason, the light has the characteristic of propagating only in thecertain directions. A typical example is reflection at an interface.Reflected light from, for example, a side wall of a cell that isparallel to incident light constructively interferes forward only anddestructively interferes in other directions, for example, backward,which results in the side wall disappearing from an image when the cellis observed with a reflection microscope. The signal intensity of thelight derived from χ⁽¹⁾ is greater than that of the light derived fromχ⁽³⁾. A typical microscope which makes a χ⁽¹⁾ distribution visible is abright field microscope and the cut-off frequency that is an indicatorof resolution is 2 NA/λ. Since the cut-off frequency of the confocalfluorescence microscope is 4 NA/λ, when the light derived from χ⁽¹⁾ isobserved with a confocal microscope, the resolution of the confocalmicroscope is lower than that of a confocal fluorescence microscope.

In contrast, for the light derived from χ⁽³⁾, there is not coherenceamong scattered beams of light, and a phase and a line width aredifferent from those of the excitation light. The light derived fromχ⁽³⁾ has a spectral width which depends on a phase relaxation time T₂,and as understood from the comparison between (A) and (C) of FIG. 2, thespectral width is wider than the line width of the excitation light fromthe laser light source 110. Since the light derived from χ⁽³⁾ which isgenerated through spontaneous process does not have coherence, it isincoherent light which does not interfere mutually. A typical example isRayleigh scattered light/fluorescence. Whichever of optical process ofRayleigh scattered light and optical process of the fluorescence is usedfor a microscope, a similar imaging characteristic is exhibited. Similarto fluorescence, the Rayleigh scattered light is radiatedomnidirectionally regardless of the incident direction of the excitationlight. Even if, for example, a side wall of a cell is parallel toincident light, the Rayleigh scattered light generated from the sidewall is radiated omnidirectionally since it is incoherent. Since thelight returns backward as well, the side wall of the cell can beobserved even if a reflection microscope is used. When the light derivedfrom χ⁽³⁾ is observed with a confocal microscope, the confocalmicroscope has the sectioning capability. Also, since the theoreticallimit value of the cut-off frequency that is an indicator of resolutionis 4 NA/λ, the resolution is equivalent to that of a confocalfluorescence microscope.

The light derived from χ⁽³⁾ includes Rayleigh scattered light. Inaddition to the Rayleigh scattered light, the light derived from χ⁽³⁾includes light generated through a spontaneous process, for example,Rayleigh-Wing scattered light, Brillouin scattered light, Ramanscattered light and fluorescence, and the intensity of the Rayleighscattered light is higher than that of the Raman scattered light or theBrillouin scattered light. A center wavelength of the Rayleigh scatteredlight included in the light derived from χ⁽³⁾ is not different from thatof the excitation light, while a center wavelength of the other lightincluded in the light derived from χ⁽³⁾ that is generated through aspontaneous process is different from that of the excitation light. Forexample, the second section of the known document, “Heitler experimentin J association” in Bussei Kenkyu (1997), 67 (4) written by KoheiMatsuda indicates that the Rayleigh scattered light is light derivedfrom χ⁽³⁾ as follows. That is, although in general, the third-ordernon-linear susceptibility χ⁽³⁾ of a substance interacting with light ismade up of a sum of multiple terms, the term corresponding to theRayleigh scattering is only one term corresponding to the diagram shownin FIG. 3 and the imaginary part of the term represents the Rayleighscattering spectrum.

Also, the ninth section of Nonlinear Optics (3rd edition) (ACADEMICPRESS) written by Robert W. Boyd indicates that the spectral width ofRayleigh scattering depends on the disturbance relaxation time forrelaxing disturbance of a density distribution (corresponding to thezero-point vibration of acoustic wave) of a substance causing Rayleighscattering.

As seen from the above, the Rayleigh scattered light is light derivedfrom χ⁽³⁾, and has a finite spectral width even if excited laser lighthas an infinitely narrow line width.

Referring back to FIG. 1, the blocking unit 103 in the detecting unit102 is described specifically. The blocking unit 103 at least partiallyblocks the light derived from χ⁽¹⁾ of the Rayleigh scattered lightderived from χ⁽³⁾ and the light derived from χ⁽¹⁾ included in the lightfrom the observation target 10. This blocking is performed by utilizinginterference occurring when the two beams of light reflected by the pairof mirrors 180 are combined by the beam splitter 170. Now, details ofthe blocking method are described using FIGS. 4 and 5.

FIG. 4 is a graph showing dependency of a theoretical signal intensityof each of Rayleigh scattered light derived from χ⁽³⁾ and light derivedfrom χ⁽¹⁾ that are obtained while optical path length difference ischanged using the pair of mirrors 180, on an optical path differencelength. FIG. 5 is a graph showing dependency of signal intensity oflight actually measured in a similar way, on optical path differencelength.

In both graphs in FIGS. 4 and 5, the horizontal axis is an optical pathdifference length measured in terms of time and the vertical axis islight signal intensity. Also, in FIGS. 4 and 5, the Rayleigh scatteredlight derived from χ⁽³⁾ is shown by χ⁽³⁾ and the light derived from χ⁽¹⁾is shown by χ⁽¹⁾.

Since the light derived from χ⁽¹⁾ is coherent light, the signalintensity changes periodically due to the interference effect as theoptical path difference length becomes longer, as shown in FIG. 4. Thespectral width of the light derived from χ⁽¹⁾ is equal to the line widthof the laser light from the laser light source 110. Therefore, when a CWlaser light with a narrowband is used as excitation light, interferenceoccurs indefinitely due to the long coherent length of the CW laserlight even if the optical path length difference is changed, and thesignal intensity of the light derived from χ⁽¹⁾ changes periodically.

In contrast, the Rayleigh scattered light derived from χ⁽³⁾ isincoherent light. Therefore, as shown in FIG. 4, as the optical pathdifference length becomes longer, although the signal intensityperiodically changes due to a measure of interference effect, theamplitude of the periodic change becomes small due to the phaserelaxation time T₂ inherent to a substance, and eventually, the signalintensity exhibits a constant value. T₂ is proportional to thereciprocal number of a spectral width, and is expected to be up toapproximately 10 ns in terms of time. This corresponds to a distance ofup to 3 m, and the optical path difference length of that degree isrealistic for an interferometer.

Using the graph showing the dependency of the signal intensity of theactually measured light on optical path difference length shown in FIG.5, a method for at least partially blocking light derived from χ⁽¹⁾ ofRayleigh scattered light derived from χ⁽³⁾ and light derived from χ⁽¹⁾is described. At first, the optical path length difference measured interms of time is set to be longer than at least 2T₂ that is twice aslong as the phase relaxation time T₂. Within the range of this opticalpath length difference, while the signal intensity of the light derivedfrom χ⁽¹⁾ changes periodically, the signal intensity of the Rayleighscattered light derived from χ⁽³⁾ is constant, as described above. Thus,the signal intensity of the light derived from χ⁽¹⁾ can be limited to anextent capable of detecting the Rayleigh scattered light derived fromχ⁽³⁾ by setting an optical path length difference so that the signalintensity of the actually measured light becomes smallest within therange of this optical path length difference, specifically, by actuallymeasuring the light signal intensity with the movable mirror 181 of thepair of mirrors 180 being arranged at a position where the signalintensity of the actually measured light becomes smallest. With thetechnique, the resolution equivalent to that of a confocal fluorescencemicroscope can be obtained without staining.

In this way, the blocking unit 103 causes waves of the light derivedfrom χ⁽¹⁾ to weaken each other through interference by adjusting theoptical path length difference using the pair of mirrors 180, to atleast partially block the light derived from χ⁽¹⁾ of the Rayleighscattered light derived from χ⁽³⁾ and the light derived from χ⁽¹⁾. Notethat the expression herein, “cause waves of the light to weaken eachother” includes splitting the light derived from χ⁽¹⁾ having coherenceinto two beams of light having therebetween the optical path lengthdifference which leads to the two beams of light being shifted in phaseby [n+(½)]λ (n is an integer) from each other, where λ is a wavelengthof light, to cause the two beams of light to cancel each other when theyare combined.

As described above, the observation apparatus 100 having theepi-illumination-type confocal optical system 101 in this embodiment atleast partially blocks the light derived from χ⁽¹⁾ and detects theRayleigh scattered light derived from χ⁽³⁾, where the Rayleigh scatteredlight derived from χ⁽³⁾ and the light derived from χ⁽¹⁾ are included inthe light obtained from the observation target irradiated withexcitation light, and outputs an observation image based on the Rayleighscattered light derived from χ⁽³⁾. In this way, without staining of theobservation target 10, the signals have sufficient intensity whileinteracting incoherently, the sectioning capability is provided, and theresolution equivalent to that of a confocal fluorescence microscope canbe obtained. The observation apparatus 100 is realized by slightlychanging a signal detecting unit in a common confocal microscope using aCW laser as the laser light source 110, and since high-speed drawing ispossible, it is suitable for quick pathological diagnosis. Note that theconfiguration in the observation apparatus 100 may be an option of theconfocal fluorescence microscope.

FIG. 6 is a schematic view of an observation apparatus 200. Since theobservation apparatus 200 has the same configuration as the observationapparatus 100 has, except that the blocking unit 103 has a lock-indetecting unit 193 and the output unit 191 in the detecting unit 102 iselectrically connected to the detector 190 via the lock-in detectingunit 193, overlapping description is omitted.

The lock-in detecting unit 193 in the blocking unit 103 is a circuitwhich performs lock-in detection of a particular light signal and iselectrically connected to the detector 190. The blocking unit 103receives the Rayleigh scattered light derived from χ⁽³⁾ and the lightderived from χ⁽¹⁾, and at least partially blocks the light derived fromχ⁽¹⁾ and detects the Rayleigh scattered light derived from χ⁽³⁾ by usingthe lock-in detecting unit 193 to perform lock-in detection of the lightfrom the observation target 10 detected by the detector 190, and detectand perform operations on a DC component and an AC component of thelight. To be specific, at first, the light derived from χ⁽¹⁾ isextracted from the light obtained from the observation target 10 byminutely vibrating, by the vibration driving unit 186, the movablemirror 181 at a high speed about a predetermined point and performingsynchronous detection at the frequency of the oscillation of the lightderived from χ⁽¹⁾. Then, the Rayleigh scattered light derived from χ⁽³⁾is detected by subtracting a half of the signal intensity of theextracted light derived from χ⁽¹⁾ from the mean signal intensity of thelight obtained from the observation target 10. In other words, the lightderived from χ⁽¹⁾ is blocked to the extent capable of detecting theRayleigh scattered light derived from χ⁽³⁾ by using the lock-indetection. Also by the technique, the resolution equivalent to that of aconfocal fluorescence microscope can be obtained without staining.

In the plurality of embodiments described above, since the observationapparatus is not of transmission type but of reflection type,observation can be conducted with a tissue cut or without cutting atissue.

In the plurality of embodiments described above, scanning is conductedusing the X-Y scanning mirror without changing the position of theobservation target. Alternatively, scanning may be conducted by changingthe position of a stage on which the observation target is placed in theX-Y direction. Also, with an additional Z scanning, the observationtarget may be measured in three dimensions by joining sectionedhigh-resolution images together in the depth direction.

In the plurality of embodiments described above, in the observationapparatus, the bandpass filter is not arranged within the detecting unitbut on the optical path in the confocal optical system. Alternatively,the bandpass filter may not be arranged in the confocal optical systembut on the optical path in the detecting unit, for example, it may bearranged in front of the detector. The configuration can also preventRaman scattered light or intrinsic fluorescence from entering into thedetector.

In the plurality of embodiments described above, the observationapparatus is described as being configured to detect Rayleigh scatteredlight derived from χ⁽³⁾ of the light obtained from the observationtarget irradiated with excitation light and output the observation imagebased on the Rayleigh scattered light derived from χ⁽³⁾. Additionally oralternatively, the observation apparatus may detect other light obtainedfrom the observation target that is derived from χ⁽³⁾ and generatedthrough a spontaneous process, for example, at least one ofRayleigh-Wing scattered light, Brillouin scattered light, Ramanscattered light and fluorescence that is light derived from χ⁽³⁾, andoutput an observation image based on the light derived from χ⁽³⁾. Inthis case, the above-described bandpass filter may not be provided onthe optical path, or other bandpass filter which transmits desired lightonly and filters out light other than the desired light may be providedon the optical path.

Also, additionally, in the plurality of embodiments described above, themicroscope may be a setting-type microscope in which the observationtarget is held sandwiched between cover glasses. In this case, it ispreferable that the cover glass be pressed so that the observationtarget is flattened. In this way, change in refraction index due to airsurrounding the observation target can be suppressed.

Also, additionally, in the plurality of embodiments described above, themovable mirror of the pair of mirrors in the blocking unit may beshifted to obtain, at a video rate, images based on the light derivedfrom χ⁽¹⁾ which has overwhelmingly higher signal intensity than thelight derived from χ⁽³⁾ has.

While the embodiments of the present invention have been described, thetechnical scope of the invention is not limited to the above describedembodiments. It is apparent to persons skilled in the art that variousalterations and improvements can be added to the above-describedembodiments. It is also apparent from the scope of the claims that theembodiments added with such alterations or improvements can be includedin the technical scope of the invention.

The operations, procedures, steps, and stages of each process performedby an apparatus, system, program, and method shown in the claims,embodiments, or diagrams can be performed in any order as long as theorder is not indicated by “prior to,” “before,” or the like and as longas the output from a previous process is not used in a later process.Even if the process flow is described using phrases such as “at first”or “next” in the claims, embodiments, or diagrams, it does notnecessarily mean that the process must be performed in this order.

EXPLANATION OF REFERENCES

10: observation target, 100: observation apparatus, 101: confocaloptical system, 102: detecting unit, 103: blocking unit, 104:controlling unit, 110: laser light source, 121, 123, 125, 127: lens,131, 133, 135: pinhole, 140: half-silvered mirror, 145: bandpass filter,150: pair of X-Y scanning mirrors, 151, 153: X-Y scanning mirror, 156:scanning driving unit, 160: objective lens, 170: beam splitter, 180:pair of mirrors, 181: movable mirror, 183: stationary mirror, 186:vibration driving unit, 190: detector, 191: output unit, 193: lock-indetecting unit

What is claimed is:
 1. An observation apparatus, comprising: a lightsource configured to irradiate an observation target with light; and aprocessing unit configured to generate an image based on Rayleighscattered light derived from χ⁽³⁾ included in light obtained from theobservation target.
 2. The observation apparatus according to claim 1,further comprising: a detecting unit configured to at least partiallyblock light derived from χ⁽¹⁾ and detect the Rayleigh scattered lightderived from χ⁽³⁾, the Rayleigh scattered light derived from χ⁽³⁾ andthe light derived from χ⁽¹⁾ being included in the light obtained fromthe observation target, wherein the processing unit generates an imagebased on the Rayleigh scattered light derived from χ⁽³⁾ detected by thedetecting unit.
 3. An observation apparatus, comprising: a light sourceconfigured to irradiate an observation target with light; and adetecting unit configured to at least partially block light derived fromχ⁽¹⁾ and detect Rayleigh scattered light derived from χ⁽³⁾, the Rayleighscattered light derived from χ⁽³⁾ and the light derived from χ⁽¹⁾ beingincluded in the light obtained from the observation target.
 4. Theobservation apparatus according to claim 2, wherein the detecting unitat least partially blocks the light derived from χ⁽¹⁾ by reducingintensity of the light derived from χ⁽¹⁾.
 5. The observation apparatusaccording to claim 2, wherein the detecting unit has: at least one lightsplitting unit arranged on an optical path of the light obtained fromthe observation target and configured to split the light into beams oflight; and at least two mirrors capable of adjusting an optical pathlength difference between the beams of light from the light splittingunit, and the detecting unit at least partially blocks the light derivedfrom χ⁽¹⁾ by utilizing interference occurring when the beams of lightreflected by the at least two mirrors are combined.
 6. The observationapparatus according to claim 5, wherein the detecting unit at leastpartially blocks the light derived from χ⁽¹⁾ by adjusting the opticalpath length difference using the at least two mirrors to cause waves ofthe light derived from χ⁽¹⁾ to weaken each other through theinterference.
 7. The observation apparatus according to claim 5, whereinthe detecting unit further includes a lock-in detecting unit configuredto at least partially block the light derived from χ⁽¹⁾ by detecting andperforming operations on a DC component and an AC component of the lightobtained from the observation target.
 8. The observation apparatusaccording to claim 5, wherein the optical path length differencemeasured in terms of time is longer than 2T₂ that is twice as long as aphase relaxation time T₂.
 9. The observation apparatus according toclaim 1, further comprising a bandpass filter arranged on an opticalpath of the light obtained from the observation target and configured totransmit the light derived from χ⁽¹⁾, and transmit the Rayleighscattered light and filter out light other than the Rayleigh scatteredlight, the Rayleigh scattered light and the light other than theRayleigh scattered light being included in light derived from χ⁽³⁾included in the light obtained from the observation target.
 10. Anobservation method, comprising generating an image based on Rayleighscattered light derived from χ⁽³⁾ included in light obtained from anobservation target irradiated with light from a light source.
 11. Theobservation method according to claim 10, comprising at least partiallyblocking light derived from χ⁽¹⁾ and detecting the Rayleigh scatteredlight derived from χ⁽³⁾, the Rayleigh scattered light derived from χ⁽³⁾and the light derived from χ⁽¹⁾ being included in the light obtainedfrom the observation target.
 12. An observation method, comprising atleast partially blocking light derived from χ⁽¹⁾ and detecting Rayleighscattered light derived from χ⁽³⁾, the Rayleigh scattered light derivedfrom χ⁽³⁾ and the light derived from χ⁽¹⁾ being included in lightobtained from an observation target irradiated with light from a lightsource.
 13. The observation method according to claim 11, wherein thedetecting has: splitting the light obtained from the observation targetinto beams of light by using a light splitting unit; adjusting anoptical path length difference between the beams of light by at leasttwo mirrors; and at least partially blocking the light derived from χ⁽¹⁾of the Rayleigh scattered light derived from χ⁽³⁾ and the light derivedfrom χ⁽¹⁾ by utilizing interference occurring when the beams of lightreflected by the at least two mirrors are combined by the lightsplitting unit.
 14. The observation method according to claim 13,wherein the detecting includes at least partially blocking the lightderived from χ⁽¹⁾ by adjusting the optical path length difference usingthe at least two mirrors to cause waves of the light derived from χ⁽¹⁾to weaken each other through the interference.
 15. The observationmethod according to claim 13, wherein the detecting includes: vibratingat least one of the at least two mirrors in a predetermined direction;and at least partially blocking the light derived from χ⁽¹⁾ byperforming lock-in detection of the light obtained from the observationtarget, and detecting and performing operations on a DC component and anAC component of the light.
 16. The observation method according to claim13, wherein the detecting further includes setting the optical pathlength difference measured in terms of time to a time longer than 2T₂that is twice as long as a phase relaxation time T₂.
 17. The observationmethod according to claim 11, further comprising transmitting the lightderived from χ⁽¹⁾, and transmitting the Rayleigh scattered light andfiltering out light other than the Rayleigh scattered light, byarranging a bandpass filter on an optical path of the light obtainedfrom the observation target, the Rayleigh scattered light and the lightother than the Rayleigh scattered light being included in light derivedfrom χ⁽³⁾ included in the light obtained from the observation target.18. An observation apparatus, comprising: a light source configured toirradiate an observation target with excitation light; at least onelight splitting unit arranged on an optical path of light obtained fromthe observation target and configured to split the light into beams oflight; at least two mirrors capable of adjusting an optical path lengthdifference between the beams of light from the light splitting unit; anda detector configured to detect Rayleigh scattered light derived fromχ⁽³⁾ included in the beams of light reflected by the at least twomirrors, wherein the beams of light from the light splitting unit arecombined after reflected by the at least two mirrors, and the detectordetects the Rayleigh scattered light derived from χ⁽³⁾ included incombined light.
 19. The observation apparatus according to claim 18,further comprising a lock-in detecting unit configured to performlock-in detection of the light obtained from the observation target, anddetect and perform operations on a DC component and an AC component ofthe light.
 20. The observation apparatus according to claim 18, furthercomprising a bandpass filter configured to transmit the Rayleighscattered light and filter out light other than the Rayleigh scatteredlight on the optical path, the Rayleigh scattered light and the lightother than the Rayleigh scattered light being included in light derivedfrom χ⁽³⁾ included in the light.